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J Physiol. Jul 1, 2008; 586(Pt 13): 3055–3059.
Published online Apr 3, 2008. doi:  10.1113/jphysiol.2008.153221
PMCID: PMC2538773

Cytoplasmic calcium oscillations and store-operated calcium influx

Abstract

Intracellular calcium oscillations have fascinated scientists for decades. They provide an important cellular signal which, unlike most signalling mechanisms, is digitally encoded. While it is generally agreed that oscillations most frequently arise from cyclical release and re-uptake of intracellularly stored calcium, it is becoming increasingly clear that influx of calcium across the plasma membrane also plays a critical role in their maintenance and even in delivering their signal to the correct cellular locus. In this review we will discuss the role played by Ca2+ entry mechanisms in Ca2+ oscillations, and approaches to understanding the molecular nature of this Ca2+ entry pathway.

Introduction

Activation of a wide variety of plasma membrane receptors results in activation of phospholipase C and generation of the Ca2+ mobilizing messenger, inositol 1,4,5-trisphosphate (IP3) (Berridge, 1993). IP3 induces the release of Ca2+ from endoplasmic reticulum stores, which in turn leads to activation of store-operated Ca2+ channels in the plasma membrane (Parekh & Putney, 2005). These two processes are readily demonstrable with maximal or near maximal concentrations of phospholipase C-linked agonists, which induce a biphasic Ca2+ signal comprising an initial release of Ca2+ followed by a sustained phase of steady Ca2+ entry (Putney et al. 1981). However, intracellular Ca2+ ([Ca2+]i) signals resulting from lower, more physiological concentrations of agonists generally result in repetitive spikes of [Ca2+]i, sometimes referred to as Ca2+ oscillations (Thomas et al. 1996). These oscillations are ideally suited for signalling a host of cellular responses because their digital nature provides a high degree of signal-to-noise discrimination (Berridge & Galione, 1988). However, the analysis of the sources and extents of Ca2+ release and the mechanisms of associated Ca2+ entry poses problems because of the very low upstream signalling strengths. The nature and regulation of associated Ca2+ influx mechanisms are of special interest because plasma membrane channels have promise as pharmacological targets. In addition, there is evidence that Ca2+ entering through plasma membrane channels is often tightly coupled to downstream effectors, even when larger global changes in Ca2+ arise from intracellular release (Chang et al. 2006, 2007).

Mechanisms of [Ca2+]i oscillations

The most commonly encountered form of [Ca2+]i oscillations involves baseline transients or spikes (Berridge, 1990; Meyer & Stryer, 1991). Baseline spikes were first described by Woods et al. (1986) and are characterized by rapidly rising transient increases in [Ca2+]i rising from a baseline of [Ca2+]i which is generally quite close to the resting level. In most instances in which [Ca2+]i transients arise periodically from a stable baseline, increasing the agonist concentration increases the frequency of the spikes with little effect on the amplitude of the spikes.

Considerable effort and speculation has been focused on the mechanism underlying the baseline spike type of calcium oscillation. Usually baseline spiking will continue for at least a few cycles in the absence of extracellular Ca2+, and thus it is generally agreed that it represents cycles of discharge and reuptake of Ca2+ by intracellular stores. The regenerative, frequency regulated nature of Ca2+ spiking suggests minimally a negative feedback as well as an amplifier or feed forward mechanism. The discoverers of this phenomenon advocated a model according to which it is the cellular level of IP3 which rises in spikes, and the [Ca2+]i changes reflect these oscillations in IP3 (Woods et al. 1987). This model suggests regulation of Ca2+ signalling at an early step in the signalling pathway, at or prior to the formation of IP3. In a conceptually similar model for baseline spikes, also featuring spikes in the level of IP3, Meyer & Stryer (1988) proposed feed forward activation of phospholipase C by [Ca2+]i together with highly cooperative opening of intracellular Ca2+ channels by IP3, but have suggested that the inactivation of the [Ca2+]i signal might result from reuptake of Ca2+ into IP3-insensitive pools, or by a negative feed back of [Ca2+]i causing desensitization of the IP3 receptor.

The second general proposal for the mechanism of baseline [Ca2+]i spiking involves fluctuations in [Ca2+]i while cellular levels of IP3 are constant. Berridge has perhaps been the major proponent of models of Ca2+ oscillations involving regulation at the IP3 receptor level (Berridge, 1990). Such mechanisms require that the IP3 receptor has the capacity to be regulated both positively and negatively by downstream signals, presumably by Ca2+ itself. There is considerable evidence for such regulation. The IP3 receptor is known to be biphasically regulated by Ca2+; in the low cytoplasmic range of [Ca2+], as [Ca2+] is increased, the ability of IP3 to release Ca2+ is increased (Iino, 1990; Finch et al. 1991; Marshall & Taylor, 1993), while at higher [Ca2+], and with more slowly developing kinetics (see below; Finch et al. 1991) the action of IP3 is inhibited (Jean & Klee, 1986; Marshall & Taylor, 1993; for a review see Foskett et al. 2007). In addition, there may be positive regulation of the receptor by Ca2+ within the lumen of the endoplasmic reticulum, which could also serve as a limiting factor for the extent of Ca2+ release during a single spike (Parys et al. 1993; but see Missiaen et al. 1999).

Recent work from Politi et al. (2006) provided strong evidence in support of an oscillating mechanism upstream of the IP3 receptor. These investigators expressed a soluble polypeptide from the IP3 binding domain of the IP3 receptor that is known to act as an IP3 ‘buffer’ in the cytoplasm (Gaspers & Thomas, 2005). As predicted from mathematical models, buffering changes in cytoplasmic IP3 prevented Ca2+ oscillations; this is not expected if oscillations occur at constant IP3 level.

Role of Ca2+ entry in [Ca2+]i oscillations

In the absence of extracellular Ca2+, oscillations are not maintained, indicating that some mechanism of Ca2+ entry across the plasma membrane is required for their maintenance (Kawanishi et al. 1989; Lewis & Cahalan, 1989). The discharge of Ca2+ stores by IP3 is known to activate store-operated Ca2+ channels in the plasma membrane (Putney, 1986; Putney et al. 2001). However, activation of phospholipase C-linked receptors is sometimes linked to other types of Ca2+ permeable channels (Barritt, 1999), and it has been argued that with low, physiological levels of agonist, such as those that produce [Ca2+]i oscillations, these non-store-operated channels might be more important (Shuttleworth, 1999). A technical difficulty in assessing the nature of the Ca2+ entry mechanism with low levels of activation is that the predicted entry of Ca2+ will be very small. Indeed given that baseline Ca2+ is usually unchanged, the prediction is that a very small amount of entry occurs that is adequately buffered by intracellular stores. The amount of Ca2+ lost from the endoplasmic reticulum during a single oscillation appears to be a very small fraction of stored Ca2+ (Bird & Putney, 2005). Thus, regardless of the mechanism of entry that replenishes this deficit, it is likely to be very small. In an earlier review, Shuttleworth argued that in many instances, Ca2+ entry may be a primary signal that secondarily activates intracellular release, resulting in [Ca2+]i oscillations (Shuttleworth, 1999). However, as mentioned above in most instances oscillations continue for at least a few cycles in the absence of extracellular Ca2+. Also, it is sometimes possible to block both Ca2+ entry and extrusion with high concentrations of lanthanides; this then results in [Ca2+]i oscillations that persist even in the absence of extracellular Ca2+ (Sneyd et al. 2004; Bird & Putney, 2005).

We will consider the argument that the entry that supports oscillations is store-operated entry, and discuss what kinds of experimental evidence can support or refute this idea. First of all, the best evidence would be the demonstration in an oscillating cell of a current with properties similar to the well-characterized calcium-release-activated calcium current (Icrac). However, in haematopoetic cells, Icrac is of the order of −2 to −3 pA pF−1, and then only under conditions whereby it is hugely amplified by preventing Ca2+ feedback inhibition (Parekh & Putney, 2005). Even with maximal store depletion, Icrac has never been observed (or at least reported) under physiological conditions, that is with weak intracellular Ca2+ buffering and at physiological membrane potentials. In other cell types, for example in one often used model the HEK293 cell, the maximal Icrac is of the order of −0.5 pA pF−1. So measurement of Icrac, or for that matter any agonist-activated Ca2+ current (Parekh, 2007), in a cell that is oscillating (which requires minimal intracellular buffering) is highly unlikely, and thus more indirect evidence is needed to test this idea.

The first approach is to compare the pharmacological sensitivity of [Ca2+]i oscillations to that known to apply to Icrac. To this end, Bird & Putney (2005) utilized two agents known to block store-operated entry and Icrac, Gd3+ and 2-aminoethyldiphenylborate (2APB). The latter compound is known to affect a number of other channel types, but notably only blocks members of the TRPC family partially (Lievremont et al. 2005), and is reported not to block channels responsible for arachidonic acid-activated currents (Mignen et al. 2003), two candidates for non-store-operated Ca2+ entry in a number of cell types (Vazquez et al. 2004; Shuttleworth et al. 2004). Gd3+ is believed to be highly specific for store-operated channels when employed in a concentration of 1 μm or less (Broad et al. 1999). Thus, the pharmacological profile of the [Ca2+]i oscillations in HEK293 cells is that which is expected of store-operated channels, likely involving Icrac. No other known Ca2+ entry pathway shares this pharmacological profile.

The second approach is to examine the roles of specific gene products known to be involved in store-operated entry, specifically, members of the Stim and Orai gene families. Stim1 serves as the major Ca2+ sensor in the endoplasmic reticulum when Ca2+ levels fall below a critical level due to active release (Roos et al. 2005; Liou et al. 2005; Brandman et al. 2007). Stim2 appears to respond to fluctuations in endoplasmic reticulum Ca2+ near the steady-state resting level (Brandman et al. 2007). The Orai (also known as CRACM) proteins function as pore-forming subunits of the store operated CRAC channels (Feske et al. 2006; Vig et al. 2006b; Zhang et al. 2006; Prakriya et al. 2006; Yeromin et al. 2006; Vig et al. 2006a). In HEK293 cells, knockdown by RNAi of either Stim1 or Orai1 results in almost complete abrogation of muscarinic receptor-linked [Ca2+]i oscillations (Wedel et al. 2007). This result is consistent with the pharmacological data and with the conclusion that in this cell line, the Ca2+ entry that supports [Ca2+]i oscillations comes through store-operated CRAC channels. One other channel has been shown to depend upon both Stim1 and Orai proteins, the arachidonic acid gated channels underlying the arachidonate-regulated calcium current, Iarc (Mignen et al. 2007, 2008). However, the function of Stim1 in Iarc is clearly not related to store depletion because it appears to be specifically Stim1 in the plasma membrane that is required (Mignen et al. 2007). Thus, it is significant that in the earlier study of the roles of these proteins in HEK293 cell [Ca2+]i oscillations, it was demonstrated that the effects of knockdown of Stim1 by RNAi could be rescued by an N-terminal fluorescent protein-tagged Stim1 (Wedel et al. 2007), which distributes only to endoplasmic reticulum and does not reach the plasma membrane (Mercer et al. 2006; Hauser & Tsien, 2007). Thus it is clear that the role of Stim1 in supporting [Ca2+]i oscillations in HEK293 cells involves its action in the endoplasmic reticulum where it functions as the calcium sensing initiator of store-operated Ca2+ entry.

The major point to be taken from this short review, as well as from previous studies from the authors' laboratory, is that a combination of carefully controlled pharmacological and molecular biological studies can provide clear and incontrovertible evidence on the role of store-operated Ca2+ entry in the Ca2+ signalling that occurs during modest and physiological strengths of cell activation. However strong this conclusion may be for the HEK293 cell line, this of course does not assure, or even suggest, that other Ca2+ mechanisms will not have major roles in other cell types. But hopefully these studies establish strategies and criteria for establishing whether, or to what degree store-operated entry contributes to the process of Ca2+ oscillations, or for that matter to any important physiological end point.

Acknowledgments

Research described in this report was supported in part by the Intramural Program of the National Institutes of Health (NIH).

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